Figure 1: In vivo transcription and protein–protein interactions of Hrp proteins. | Nature Communications

Figure 1: In vivo transcription and protein–protein interactions of Hrp proteins.

From: Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity

Figure 1

(a) Domain organization of EBPs showing the seven conserved regions (C1–C7) and their associated functions. HrpR and HrpS key motif sequences are given with the sites of amino-acid substitutions (used in c, d, e below) indicated (black typeface numbering refers to amino-acid position in either HrpR or HrpS). (b) In vivo transcription activation activities measured from the chromosomal promoter hrpL∷lacZ reporter construct—given as a percentage of WT co-expressed (single operon) HrpRS activity (where WT=100%)—of singly expressed HrpR or HrpS, and co-expressed HrpR+HrpS (HrpR+S) (from different plasmids). HrpV expression abolishes HrpRS in vivo activity (HrpRS+HrpV), which is partly relieved when co-expressing HrpG (HrpRS+HrpV+HrpG). (c) As in b transcription activation by HrpRS, where HrpR carries mutations as indicated. (d) As in b transcription activation by HrpRS, where HrpS carries mutations as indicated. (e) As in b transcription activation by HrpRS, where HrpS carries the gain of function mutation Y85F. (f) Bar graph depicting results of the BACTH protein–protein binding interactions between HrpR (black), HrpS (light grey), HrpS1−275 (dark grey), HrpV (white), HrpG (grey) inferred by β-galactosidase production and displayed in Miller Units (MU)32. B/ground represents the background level of β-galactosidase activity detected for the negative control (30 MU—obtained with the two-hybrid vectors in the absence of protein fusions). (g) Scheme of the expression construct for the BACTH three component system. (h) Bar graph depicting the binding interactions between T25-fused proteins HrpR, HrpS, HrpV and HrpG with T18C-fused HrpS in the presence of co-expressed HrpV in a three-component two-hybrid system35 inferred by β-galactosidase production and displayed in Miller Units. In b–e, all assays were minimally performed in triplicate and standard errors of the mean are shown. Protein expression was verified by western blotting (see Supplementary Fig. S3).

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